The present invention relates to an apparatus and a method for detecting the presence of an analyte in a sample obtained from a person or animal. The present invention is applicable to the detection of different types of pathogens and particularly to the detection of an influenza virus.
Influenza is a disease that is caused in the animal species by the influenza virus. In humans, infection by the influenza virus may cause symptoms that are also common to less serious diseases such as the common cold and/or that are attributed to other diseases, such as, for example, gastroenteritis. Thus, infection with the influenza virus may be difficult to diagnose. If undiagnosed or wrongly diagnosed, influenza may lead to more serious health consequences, especially for certain age-groups, particularly the very young and the elderly, and/or for those suffering from other chronic medical conditions.
Influenza is transmittable from infected mammals via different media, for example, bodily fluids that are propagated via air by coughing or sneezing or contact with the blood or faeces of an infected person or animal via a contaminated surface.
Its contagious nature exacerbates the spread of influenza, which typically peaks during the winter months. When the influenza virus spreads rapidly in the human/animal population in a locality, this is referred to as an epidemic. When it spreads over a larger geographical area, for example, a country, a continent, or even across several continents, this is referred to as a pandemic. Controlling pandemics is a challenge since a virus strain found in a particular animal species, for example, birds, may mutate into a virulent form and spread amongst the population of that animal species if left uncontrolled and may kill large numbers of that population. The aforesaid mutated virus strain may cross-over into a second animal species, for example, humans, by interaction with a virus strain found in that second animal species to form an evolved version of the mutated virus strain, which can further mutate and cause a pandemic in the second animal species if not contained.
The types of known influenza virus types include: influenza virus A and its sub-types, influenza virus 8 and influenza virus C. It is the influenza virus A and its sub-types that are the most virulent and attributed to causing pandemics. The most recently identified sub-type of the influenza virus A, which potentially poses a pandemic threat is H5N1, more commonly known as the avian flu virus. Virus sub-types are also referred to as strains.
In order to reduce the spread of the influenza virus and/or the outbreak of a pandemic, infected persons should ideally be identified by the detection of the virus. If this is done early enough, for example, within 48 hours of infection, then the fatal consequences of the virus on an infected person may be reduced by the administration of appropriate drugs and, importantly, measures may be taken so that the infected person does not further spread the virus to others. Apart from also facilitating the surveillance of a virus outbreak, identification of an infected person may also assist in the development of a vaccine to reduce the spreading of the virus since vaccines contain inactivated forms of the most recently-detected influenza strain. Due to the mutative ability of viruses, it is typically the case that vaccines developed to contain a recent influenza outbreak may not be suitable for the same purpose for a subsequent influenza outbreak, for example, in the following year.
Some of the techniques that have been applied for influenza virus testing have been discussed herebelow.
Real-Time Polymerase Chain Reaction (RT-PCR)
In RT-PCR, a sample collected from the throat and/or nasopharynx of a person/animal is subjected to pre-treatment, thereby to further increase the solubility of mucus contained in the sample. The pre-treated sample is then processed so that a specific portion of the genome of a virus present in the sample, particularly, the deoxyribonucleic acid (DNA) or the ribonucleic acid (RNA), is amplified and rendered readable. The latter is done by using a complementary set of primers that is known to be associated to a specific section of the DNA/RNA of the virus that is being tested for.
RT-PCR is considered to be one of the techniques of choice for influenza virus testing on account of reliability and duration since it takes under 5 hours to obtain results. However, it has some associated disadvantages. Processing of the sample such as amplification of the DNA/RNA is specialized and done with the aid of peripheral equipment, which factors mean that such tests are done by skilled personnel and are expensive to perform. RT-PCR is a laboratory-based technique and may not be suitable for on-field testing, that is, on a site where infection by an influenza virus is considered to have occurred. Although it has recently been proposed to facilitate on-field detection by using a lab-on chip concept for RT-PCR, that is by incorporating all the components of the RT-PCR test on a common platform/substrate that can be transported to a site where testing is to be conducted, this may be expensive and testing can, again, only be performed by trained staff.
Agglutination Testing
This is a serological test which uses as its basis the fact that the influenza virus causes red blood cells to agglutinate or clump together. By subjecting a blood sample to a known antibody, the presence of a virus can be detected since the antibody would inhibit the agglutination of red blood cells by the virus. The agglutination inhibition can be observed by the sedimentation of red blood cells into the bottom of a conical test well to produce a distinct red dot. By respectively subjecting blood samples to increasingly diluted concentrations of the antibody, the sedimentation is seen to progressively decrease. The last dilution for which agglutination inhibition is detected provides information on the virus concentration in the host.
Like RT-PCR, agglutination testing can only be performed by trained staff and is done using specialized equipment and/or reagents. Furthermore, the cost and stability of reagents are an issue. This technique is primarily suited to being conducted in a laboratory; it would generally be considered to be unsuitable for on-field testing due to possible health and safety issues that could arise with open platforms. Due to the possible inconsistency in the way that the testing is conducted and/or interpretation of the results, agglutination testing may not be acceptable for surveillance of virus outbreaks. It is generally used for research and/or monitoring the health of individuals.
Tests Based on Immunoassays
Such testing is based on detecting viruses or antibodies produced in a host in response to infection by a virus by detecting antibody/antigen binding.
For antigen/antibody detection, immunofluorescence can be used. In this case, particles that are known to tag to specific antigens or antibodies and that fluoresce when illuminated with specific wavelengths of light are used. Although this technique can be used for detecting the presence of an influenza virus and for the further sub-typing of the influenza virus A, specialized microscopic techniques and/or reagents that are operable by skilled staff make this technique expensive, fragile and render it unsuitable for on-field virus testing.
There are some commercially available diagnostic tests for the detection of influenza viruses in a time-scale of between 10 to 30 minutes. They are based on lateral flow immunochromatography and are embodied in the form of strip-type detectors that provide a binary read-out on virus detection. Such tests may vary in the types of influenza viruses that are detectable and whether influenza types can be distinguished. Currently available tests are categorized based on the detection of: only influenza A viruses; both influenza A and B viruses, but no distinction can be made between the two types, or both influenza A and B and distinction can be made between the two. Currently, such diagnostic testing kits are unable to provide information on influenza A subtypes, which are attributed with causing/potentially causing pandemics. Furthermore, there is inconsistency in the sample specimens that are used for such tests, for example, some may use throat, nasopharyngeal, or nasal aspirates whilst others may use swabs, or washes. The specificity and, in particular, the sensitivity of such tests are lower than for viral culture and variable according to the particular test. Although the results of such tests may be obtained on a much shorter time-scale compared to other known techniques for influenza testing, a trade-off exists with respect to the sensitivity. Due to the lower sensitivity, negative test results may need to be confirmed by viral cultures or other methods such as agglutination testing or RT-PCR in order to flag whether they are false-negative results, especially during periods of peak community influenza activity. In contrast, false-positive rapid test results are less likely, but can occur during periods of low influenza activity.
U.S. Pat. No. 5,723,345 discloses a method of determining the amount of a substance in a liquid sample comprising: flowing a signal substance generator and a liquid sample through a predetermined channel in a predetermined direction, such that a specific binding reaction takes place with at least the substance and the signal substance generator, thereby causing the formation of a specific distribution of the signal substance generator in the channel, which is dependent on the concentration of the substance, the specific distribution formed by an affinity chromatographic, or an immunoprecipitation process; generating a signal substance from the signal substance generator specifically distributed in the channel; allowing dispersion of unreacted signal substance generator throughout the channel; allowing diffusion of the signal substance to a plurality of detection means arranged in different positions in the flow direction, detecting the signal substance with the plurality of detection means, and determining the concentration of the substance from the relative signal detected at the detection means. In this method, several different types of reagents and chemicals are used, which may increase the cost of producing such devices. Further, signal acquisition devices and signal-processing using mathematical models may serve to increase the complexity of operating this device. This requires adding functionality to the device, which increases its cost of fabrication and may serve to reduce the overall reliability, stability, and shelf lifetime of the device.
US-A1-2003/0045001 discloses an immunochromatographic test strip having a curved sample application zone, which functions similarly to standard immunochromatographic test strips but has a different sample collection zone. This method may ease the collection of some kinds of samples, such as, for example, blood taken from a finger, but may not be suitable for loading typical samples for detecting influenza viruses and other pathogens onto a test strip. Being based on a similar principle to immunochromatographic strip testing, the present technique may also suffer from the same drawbacks.
Accordingly, it is desirable to provide a technique for the detection of an influenza virus that mitigates and/or obviates the drawbacks associated to known techniques for the same purpose.